Senile amyloid plaques are one of the diagnostic hallmarks of Alzheimer's disease (AD). However, the severity of clinical symptoms of AD is weakly correlated with the plaque load. AD symptoms severity is reported to be more strongly correlated with the level of soluble amyloid-ß (Aß) assemblies. Formation of soluble Aß assemblies is stimulated by monomeric Aß accumulation in the brain, which has been related to its faulty cerebral clearance. Studies tend to focus on the neurotoxicity of specific Aß species. There are relatively few studies investigating toxic effects of Aß on the endothelial cells of the blood-brain barrier (BBB). We hypothesized that a soluble Aß pool more closely resembling the in vivo situation composed of a mixture of Aß40 monomer and Aß42 oligomer would exert higher toxicity against hCMEC/D3 cells as an in vitro BBB model than either component alone. We observed that, in addition to a disruptive effect on the endothelial cells integrity due to enhancement of the paracellular permeability of the hCMEC/D3 monolayer, the Aß mixture significantly decreased monomeric Aß transport across the cell culture model. Consistent with its effect on Aß transport, Aß mixture treatment for 24h resulted in LRP1 down-regulation and RAGE up-regulation in hCMEC/D3 cells. The individual Aß species separately failed to alter Aß clearance or the cell-based BBB model integrity. Our study offers, for the first time, evidence that a mixture of soluble Aß species, at nanomolar concentrations, disrupts endothelial cells integrity and its own transport across an in vitro model of the BBB.